Acta Neurol. Scandinav. 52, 71-80, 1975
Department of Neurology and Neuromuscular Research Lahoratory, Mayo Clinic and Mayo Foundation, Rochester, Minnesota, U.S.A.
EFFECTS O F MICROEMBOLIZATION ON THE SKELETAL MUSCLE BLOOD FLOW
A Critique of the Microvascular Occlusion Model of Duchenne Dystrophy GUDRUNBOYSEN and ANDREWG. ENGEL ABSTRACT Muscle blood flow (MBF) during exercise was determined i n both anterior tibia1 muscles of 12 rabbits by the local l33Xe method before and after emholization of 20 to 80 #m dextran particles into the right femoral artery. With a relatively low dose of particles (dry weight: 8.7 mg), MBF was significantly decreased up to 4 days after each embolization, and mild histologic abnormalities were observed in the embolized muscles. A four-fold higher dose resulted i n a marked and permanent decrease of t h e MBF, ischemic necrosis of the distal leg muscles and toe gangrene. In t h e light of these findings and the previously demonstrated normal MBF i n Duchenne dystrophy, the validity of t h e assumption t h a t Duchenne dystrophy is caused hy small vessel occlusions is questioned.
An experimental model of Duchenne muscular dystrophy based on microarterial embolization with dextran particles in rabbits has been reported (Hathaway et al. 1970). One to 12 weeks after 2 or 3 repeated embolizations 2 to 4 weeks apart, a histologic picture in muscle resembling Duchenne dystrophy was found. More recent studies, however, have questioned that microvascular disease is the primary pathogenetic factor in Duchenne dystrophy. I n patients with this disease, the muscle blood flow, as determined by the intramuscular 133Xe method, is not decreased in the affected extremities (Paulson et al. 1974). Although replication of muscle capillary basement membranes can be found in Duchenne dystrophy, necrotic capillaries are not This work was supported i n part by a Research Center Grant of t h e Muscular Dystrophy Association of America.
72 present, the mean muscle fiber volume served per capillary is normal and the average capillary diameter is slightly larger than normal (Jerusalem et al. 1974). In this study we wished to test the assumption that experimental microvascular occlusive disease represent a valid model for Duchenne dystrophy. F o r this reason we examined the effects of small vessel embolization on the muscle blood flow (MBF). The washout of intramuscularly deposited 133Xe was used in estimating MBF during a standardized exercise, as this method has been previously employed in detecting arterial occlusive disease (Lassen et al. 1964, Alperf e t al. 1968, Larsen 1973). If this method failed to show any decrease in M R F after microarterial occlusion, the normal M B F in human Duchenne dystrophy would not be incompatible with the microvascular occlusion hypothesis. On the other hand, if experimental microvascular occlusion did decrease the MBF, the applicability of the hypothesis to human Duchenne dystrophy would be open to question.
METHODS Twelve white rabbits of either sex and with initial body weights of 3.5 to 1.5 kg were used. The animals were anesthetized with 100-130 mg pentobarbital intravenously, and this dose was subsequently supplemented as required. Blood flow was determined i n both right and left anterior tibia1 muscles of all animals hy the intramuscular l33Xe method (Lassen e f al. 1964, Tsnnesen & Sejrsen 1970). Using a 27 gauge needle, 100-200 &i of l33Xe dissolved in 0.04-0.08 ml saline was deposited in the distal third of the muscle. Tracer washout was monitored with a widely collimated NaI crystal scintillation detector connected to a ratemeter; and, after Iogarithmic conversion, the clearance curve was pIotted with a linear recorder. The initial count rate was ahout 105 counts per minute, with a background activity less than 0.1 per cent of the initial count rate. Following the deposition of the isotope, the radioactivity was followed for a sufficient length of time (usually 10-15 min) for the initial hyperemia due to the trauma of the injection to subside. The common peroneal nerve was then stimulated percutaneously with 3 per sec supramaximal square-wave pulses for 3 min. This usually produced strong and rhythmic dorsiflexions of the foot. The initial and steepest part of the laXe washout curve observed during the period of stimulation was used for flow calculations according to the formula :
MBF
= 0.60
?(
2.3 X D m1/100 g/min,
where 0.60 is the muscle to blood partition coefficient for i33Xe a t a hematocrit level of 40 per cent ( T s n n e s e n & Sejrsen 1970) ; 2.3 is the conversion factor from base 10 to natural logarithm; and D the slope of the clearance curve i n p e r cent of a decade per minute. Since the muscle bIood flow i n either Jimb might he affected by the level of anesthesia, variations in blood pressure, levels of circulating vasoactive amines (Shephard 1963, B a l k a n e t al. 1965), and probably by other factors as well, all flow
measurements were done bilaterally i n every animal, the unembolized left leg of a given animal serving as a matched control for t h e right leg of the same animal. Flow measurements obtained i n embolized and control legs of each animal were compared by the paired t-test. Following a first set of MBF measurements in both legs, the right femoral artery was exposed at the groin. Spherical dextran particles, 20 to 80 pm in diameter in the dry state t Sephadex G-25 @ fine, Pharmacia), were injected through a 23 gauge siliconized needle connected by a short flexible catheter ~“Butterfly-23”@infusion set, Abbott) to a syringe containing the particles suspended in saline. The needle and catheter were prefilled with saline to avoid blockage of the needle by the particles prior to injection. To ensure injection of any dextran particles that may have sedimented in the syringe o r infusion set during the initial injection, the syringe and infusion set were next flushed with 4 m l saline. After removal of the needle, hemostasis was effected within a few minutes by compression of the femoral artery i n the groin. When a good pulse was palpable in the right saphenous artery on the medial side of the knee, MBF was again determined in both anterior tibial muscles. MBF measurements were repeated on days 1 ( 5 animals), 2 (6 animals), and 4 (4 animals) after embolization. Five animals had a second embolization 2 weeks, and one animal a third embolization 4 weeks after the first embolization; each time with repetition of the MBF procedures. The Sephadex suspension was prepared by suspending 1 ml (870 mg) of dry dextran particles in 100 ml of normal saline. In 10 animals all embolizations were done with 1 ml (containing 8.7 mg of the dry particles of this suspension, while 2 animals had a single injection of 4 ml of t h e same suspension. Muscle biopsies obtained a t selected intervals after embolization from proximal portions of both anterior tibial muscles and from both gastrocnemius and rectus femoris muscles were quick-frozen for histochemical studies. These were carried out according to previously described methods ( E n g e l & Dale 1968).
RESULTS
Muscle Blood Flow. The MBF data are shown in Table 1 and Figure 1. Before embolization, MBF during exercise ranged between 45 and 80 m1/100 g/niin. The mean value for the left side (59.3) was essentially identical with that for the right side (58.1), and the two values were not significantly different by the paired t-test. Following the embolization of 1 ml of dextran particles into the right femoral artery, the exercise MBF decreased in both anterior tibial muscles; but the decrease in the injected leg (mean MBF: 12.6 m1/100 g/min) was consistently more marked than in the uninjected control leg (mean MBF: 39.1 m1/100 g/min) ; and the two means were significantly different by the paired t-test. On days 1 and 2 after embolization, the MBF was still significantly lower in the embolized than in the control legs. By day 4, the mean MBF on the embolized side was 75 per cent of that on the control side, and the two means were no longer significantly different. On day 14, no
E-C
1.17 5.04 NS
E-C
Department of Neurology and Neuromuscular Research Lahoratory, Mayo Clinic and Mayo Foundation, Rochester, Minnesota, U.S.A.
EFFECTS O F MICROEMBOLIZATION ON THE SKELETAL MUSCLE BLOOD FLOW
A Critique of the Microvascular Occlusion Model of Duchenne Dystrophy GUDRUNBOYSEN and ANDREWG. ENGEL ABSTRACT Muscle blood flow (MBF) during exercise was determined i n both anterior tibia1 muscles of 12 rabbits by the local l33Xe method before and after emholization of 20 to 80 #m dextran particles into the right femoral artery. With a relatively low dose of particles (dry weight: 8.7 mg), MBF was significantly decreased up to 4 days after each embolization, and mild histologic abnormalities were observed in the embolized muscles. A four-fold higher dose resulted i n a marked and permanent decrease of t h e MBF, ischemic necrosis of the distal leg muscles and toe gangrene. In t h e light of these findings and the previously demonstrated normal MBF i n Duchenne dystrophy, the validity of t h e assumption t h a t Duchenne dystrophy is caused hy small vessel occlusions is questioned.
An experimental model of Duchenne muscular dystrophy based on microarterial embolization with dextran particles in rabbits has been reported (Hathaway et al. 1970). One to 12 weeks after 2 or 3 repeated embolizations 2 to 4 weeks apart, a histologic picture in muscle resembling Duchenne dystrophy was found. More recent studies, however, have questioned that microvascular disease is the primary pathogenetic factor in Duchenne dystrophy. I n patients with this disease, the muscle blood flow, as determined by the intramuscular 133Xe method, is not decreased in the affected extremities (Paulson et al. 1974). Although replication of muscle capillary basement membranes can be found in Duchenne dystrophy, necrotic capillaries are not This work was supported i n part by a Research Center Grant of t h e Muscular Dystrophy Association of America.
72 present, the mean muscle fiber volume served per capillary is normal and the average capillary diameter is slightly larger than normal (Jerusalem et al. 1974). In this study we wished to test the assumption that experimental microvascular occlusive disease represent a valid model for Duchenne dystrophy. F o r this reason we examined the effects of small vessel embolization on the muscle blood flow (MBF). The washout of intramuscularly deposited 133Xe was used in estimating MBF during a standardized exercise, as this method has been previously employed in detecting arterial occlusive disease (Lassen et al. 1964, Alperf e t al. 1968, Larsen 1973). If this method failed to show any decrease in M R F after microarterial occlusion, the normal M B F in human Duchenne dystrophy would not be incompatible with the microvascular occlusion hypothesis. On the other hand, if experimental microvascular occlusion did decrease the MBF, the applicability of the hypothesis to human Duchenne dystrophy would be open to question.
METHODS Twelve white rabbits of either sex and with initial body weights of 3.5 to 1.5 kg were used. The animals were anesthetized with 100-130 mg pentobarbital intravenously, and this dose was subsequently supplemented as required. Blood flow was determined i n both right and left anterior tibia1 muscles of all animals hy the intramuscular l33Xe method (Lassen e f al. 1964, Tsnnesen & Sejrsen 1970). Using a 27 gauge needle, 100-200 &i of l33Xe dissolved in 0.04-0.08 ml saline was deposited in the distal third of the muscle. Tracer washout was monitored with a widely collimated NaI crystal scintillation detector connected to a ratemeter; and, after Iogarithmic conversion, the clearance curve was pIotted with a linear recorder. The initial count rate was ahout 105 counts per minute, with a background activity less than 0.1 per cent of the initial count rate. Following the deposition of the isotope, the radioactivity was followed for a sufficient length of time (usually 10-15 min) for the initial hyperemia due to the trauma of the injection to subside. The common peroneal nerve was then stimulated percutaneously with 3 per sec supramaximal square-wave pulses for 3 min. This usually produced strong and rhythmic dorsiflexions of the foot. The initial and steepest part of the laXe washout curve observed during the period of stimulation was used for flow calculations according to the formula :
MBF
= 0.60
?(
2.3 X D m1/100 g/min,
where 0.60 is the muscle to blood partition coefficient for i33Xe a t a hematocrit level of 40 per cent ( T s n n e s e n & Sejrsen 1970) ; 2.3 is the conversion factor from base 10 to natural logarithm; and D the slope of the clearance curve i n p e r cent of a decade per minute. Since the muscle bIood flow i n either Jimb might he affected by the level of anesthesia, variations in blood pressure, levels of circulating vasoactive amines (Shephard 1963, B a l k a n e t al. 1965), and probably by other factors as well, all flow
measurements were done bilaterally i n every animal, the unembolized left leg of a given animal serving as a matched control for t h e right leg of the same animal. Flow measurements obtained i n embolized and control legs of each animal were compared by the paired t-test. Following a first set of MBF measurements in both legs, the right femoral artery was exposed at the groin. Spherical dextran particles, 20 to 80 pm in diameter in the dry state t Sephadex G-25 @ fine, Pharmacia), were injected through a 23 gauge siliconized needle connected by a short flexible catheter ~“Butterfly-23”@infusion set, Abbott) to a syringe containing the particles suspended in saline. The needle and catheter were prefilled with saline to avoid blockage of the needle by the particles prior to injection. To ensure injection of any dextran particles that may have sedimented in the syringe o r infusion set during the initial injection, the syringe and infusion set were next flushed with 4 m l saline. After removal of the needle, hemostasis was effected within a few minutes by compression of the femoral artery i n the groin. When a good pulse was palpable in the right saphenous artery on the medial side of the knee, MBF was again determined in both anterior tibial muscles. MBF measurements were repeated on days 1 ( 5 animals), 2 (6 animals), and 4 (4 animals) after embolization. Five animals had a second embolization 2 weeks, and one animal a third embolization 4 weeks after the first embolization; each time with repetition of the MBF procedures. The Sephadex suspension was prepared by suspending 1 ml (870 mg) of dry dextran particles in 100 ml of normal saline. In 10 animals all embolizations were done with 1 ml (containing 8.7 mg of the dry particles of this suspension, while 2 animals had a single injection of 4 ml of t h e same suspension. Muscle biopsies obtained a t selected intervals after embolization from proximal portions of both anterior tibial muscles and from both gastrocnemius and rectus femoris muscles were quick-frozen for histochemical studies. These were carried out according to previously described methods ( E n g e l & Dale 1968).
RESULTS
Muscle Blood Flow. The MBF data are shown in Table 1 and Figure 1. Before embolization, MBF during exercise ranged between 45 and 80 m1/100 g/niin. The mean value for the left side (59.3) was essentially identical with that for the right side (58.1), and the two values were not significantly different by the paired t-test. Following the embolization of 1 ml of dextran particles into the right femoral artery, the exercise MBF decreased in both anterior tibial muscles; but the decrease in the injected leg (mean MBF: 12.6 m1/100 g/min) was consistently more marked than in the uninjected control leg (mean MBF: 39.1 m1/100 g/min) ; and the two means were significantly different by the paired t-test. On days 1 and 2 after embolization, the MBF was still significantly lower in the embolized than in the control legs. By day 4, the mean MBF on the embolized side was 75 per cent of that on the control side, and the two means were no longer significantly different. On day 14, no
E-C
1.17 5.04 NS
E-C
